Bottom Line:
Additionally, two other signaling pathways must cooperate with p38-CHOP to effect apoptosis.One involves the type A scavenger receptor (SRA).Thus, FC-induced apoptosis requires cholesterol trafficking to the ER, which triggers p38-CHOP and JNK2, and engagement of the SRA.

ABSTRACTMacrophage death in advanced atherosclerosis promotes necrosis and plaque destabilization. A likely cause of macrophage death is accumulation of free cholesterol (FC) in the ER, leading to activation of the unfolded protein response (UPR) and C/EBP homologous protein (CHOP)-induced apoptosis. Here we show that p38 MAPK signaling is necessary for CHOP induction and apoptosis. Additionally, two other signaling pathways must cooperate with p38-CHOP to effect apoptosis. One involves the type A scavenger receptor (SRA). As evidence, FC loading by non-SRA mechanisms activates p38 and CHOP, but not apoptosis unless the SRA is engaged. The other pathway involves c-Jun NH2-terminal kinase (JNK)2, which is activated by cholesterol trafficking to the ER, but is independent of CHOP. Thus, FC-induced apoptosis requires cholesterol trafficking to the ER, which triggers p38-CHOP and JNK2, and engagement of the SRA. These findings have important implications for understanding how the UPR, MAPKs, and the SRA might conspire to cause macrophage death, lesional necrosis, and plaque destabilization in advanced atherosclerotic lesions.

fig5: FC loading using CD-cholesterol or β-VLDL induces p38 MAPK phosphorylation and UPR induction. (A) Macrophages were left untreated for 5 h or were incubated with CD-cholesterol (CD-Chol) ± 58035 for the indicated number of hours. Whole cell lysate was immunoblotted for phospho-p38, phospho-MK2, and actin. (B) Macrophages were left untreated or incubated with CD-cholesterol plus 58035 for 8 and 24 h. Nuclear extracts were isolated as described in “Materials and methods” and were subjected to immunoblot analysis for CHOP and nucleoplasmin (as a loading control). Cytosolic extracts were immunoblotted for phospho-Thr980 PERK and actin. (C) Macrophages were incubated with CD-cholesterol or 50 μg/ml β-VLDL plus 58035 for the indicated number of hours. Whole cell lysates were subjected to immunoblot analysis for CHOP, phospho-p38, and actin. (D) Macrophages were incubated with 50 μg/ml β-VLDL or 100 μg/ml ac-LDL plus 58035 for 24 h and then stained with Alexa 488 Annexin V (green) and propidium iodide (red). Bar, 25 μm.

Mentions:
To begin, we determined whether CD-cholesterol loading led to p38 phosphorylation and UPR induction. As shown in Fig. 5 A, p38 and MK2 were phosphorylated in CD-cholesterol–loaded macrophages. Early activation of p38 by CD-cholesterol (i.e., at 5–8 h) was observed independently of ACAT inhibition. This finding may be related to a direct effect of CD-derived cholesterol on the plasma membrane; this is in contrast to the situation with ac-LDL plus the ACAT inhibitor, which involves trafficking of lipoprotein-derived cholesterol to the ER (Li et al., 2005). However, activation of p38 at 24 h was dependent on ACAT inhibition, which suggested that this later phase of activation may be dependent on intracellular cholesterol trafficking. Most importantly, CD-cholesterol led to induction of CHOP and phospho-PERK (ds-RNA–activated protein kinase-like ER kinase) by 24 h, indicating activation of the UPR (Fig. 5 B). Given these data, we considered the possibility that the inability of CD-cholesterol plus the ACAT inhibitor to induce apoptosis, even at 48 h (unpublished data), was due to the later activation of the UPR. For example, it is possible that the gradual activation of the UPR by CD-cholesterol could “precondition” the cells, and thus, render them resistant to UPR-induced apoptosis, as has been observed in other systems (Hung et al., 2003), In this regard, we incubated ACAT-inhibited macrophages with β–very low density lipoprotein (VLDL), a cholesterol-rich lipoprotein that is endocytosed rapidly by macrophages. As shown in Fig. 5 C, FC loading using β-VLDL caused relatively rapid induction of CHOP and activation of p38, similar to the kinetics using ac-LDL; however, apoptosis was not induced (Fig. 5 D). CHOP induction by β-VLDL plus 58035 was diminished in p38α-deficient macrophages (Fig. S3; available at http://www.jcb.org/cgi/content/full/jcb.200502078/DC1), which demonstrated its dependence on p38 signaling. Thus, even rapid activation of CHOP and p38 is unable to induce apoptosis when a lipoprotein other than ac-LDL is used. Therefore, we considered an alternative hypothesis—that FC-induced p38/UPR activation is necessary, but not sufficient, for apoptosis, and apoptosis of UPR-activated macrophages requires another “hit” effected by ac-LDL, but not by CD-cholesterol or β-VLDL.

fig5: FC loading using CD-cholesterol or β-VLDL induces p38 MAPK phosphorylation and UPR induction. (A) Macrophages were left untreated for 5 h or were incubated with CD-cholesterol (CD-Chol) ± 58035 for the indicated number of hours. Whole cell lysate was immunoblotted for phospho-p38, phospho-MK2, and actin. (B) Macrophages were left untreated or incubated with CD-cholesterol plus 58035 for 8 and 24 h. Nuclear extracts were isolated as described in “Materials and methods” and were subjected to immunoblot analysis for CHOP and nucleoplasmin (as a loading control). Cytosolic extracts were immunoblotted for phospho-Thr980 PERK and actin. (C) Macrophages were incubated with CD-cholesterol or 50 μg/ml β-VLDL plus 58035 for the indicated number of hours. Whole cell lysates were subjected to immunoblot analysis for CHOP, phospho-p38, and actin. (D) Macrophages were incubated with 50 μg/ml β-VLDL or 100 μg/ml ac-LDL plus 58035 for 24 h and then stained with Alexa 488 Annexin V (green) and propidium iodide (red). Bar, 25 μm.

Mentions:
To begin, we determined whether CD-cholesterol loading led to p38 phosphorylation and UPR induction. As shown in Fig. 5 A, p38 and MK2 were phosphorylated in CD-cholesterol–loaded macrophages. Early activation of p38 by CD-cholesterol (i.e., at 5–8 h) was observed independently of ACAT inhibition. This finding may be related to a direct effect of CD-derived cholesterol on the plasma membrane; this is in contrast to the situation with ac-LDL plus the ACAT inhibitor, which involves trafficking of lipoprotein-derived cholesterol to the ER (Li et al., 2005). However, activation of p38 at 24 h was dependent on ACAT inhibition, which suggested that this later phase of activation may be dependent on intracellular cholesterol trafficking. Most importantly, CD-cholesterol led to induction of CHOP and phospho-PERK (ds-RNA–activated protein kinase-like ER kinase) by 24 h, indicating activation of the UPR (Fig. 5 B). Given these data, we considered the possibility that the inability of CD-cholesterol plus the ACAT inhibitor to induce apoptosis, even at 48 h (unpublished data), was due to the later activation of the UPR. For example, it is possible that the gradual activation of the UPR by CD-cholesterol could “precondition” the cells, and thus, render them resistant to UPR-induced apoptosis, as has been observed in other systems (Hung et al., 2003), In this regard, we incubated ACAT-inhibited macrophages with β–very low density lipoprotein (VLDL), a cholesterol-rich lipoprotein that is endocytosed rapidly by macrophages. As shown in Fig. 5 C, FC loading using β-VLDL caused relatively rapid induction of CHOP and activation of p38, similar to the kinetics using ac-LDL; however, apoptosis was not induced (Fig. 5 D). CHOP induction by β-VLDL plus 58035 was diminished in p38α-deficient macrophages (Fig. S3; available at http://www.jcb.org/cgi/content/full/jcb.200502078/DC1), which demonstrated its dependence on p38 signaling. Thus, even rapid activation of CHOP and p38 is unable to induce apoptosis when a lipoprotein other than ac-LDL is used. Therefore, we considered an alternative hypothesis—that FC-induced p38/UPR activation is necessary, but not sufficient, for apoptosis, and apoptosis of UPR-activated macrophages requires another “hit” effected by ac-LDL, but not by CD-cholesterol or β-VLDL.

Bottom Line:
Additionally, two other signaling pathways must cooperate with p38-CHOP to effect apoptosis.One involves the type A scavenger receptor (SRA).Thus, FC-induced apoptosis requires cholesterol trafficking to the ER, which triggers p38-CHOP and JNK2, and engagement of the SRA.

ABSTRACTMacrophage death in advanced atherosclerosis promotes necrosis and plaque destabilization. A likely cause of macrophage death is accumulation of free cholesterol (FC) in the ER, leading to activation of the unfolded protein response (UPR) and C/EBP homologous protein (CHOP)-induced apoptosis. Here we show that p38 MAPK signaling is necessary for CHOP induction and apoptosis. Additionally, two other signaling pathways must cooperate with p38-CHOP to effect apoptosis. One involves the type A scavenger receptor (SRA). As evidence, FC loading by non-SRA mechanisms activates p38 and CHOP, but not apoptosis unless the SRA is engaged. The other pathway involves c-Jun NH2-terminal kinase (JNK)2, which is activated by cholesterol trafficking to the ER, but is independent of CHOP. Thus, FC-induced apoptosis requires cholesterol trafficking to the ER, which triggers p38-CHOP and JNK2, and engagement of the SRA. These findings have important implications for understanding how the UPR, MAPKs, and the SRA might conspire to cause macrophage death, lesional necrosis, and plaque destabilization in advanced atherosclerotic lesions.